Listeria monocytogenes is a ubiquitous Gram-positive bacterium that can cause serious food-borne infections in pregnant women, newborns and immunocompromised or older adults. Some patients develop infections in the central nervous system because of the unusual ability of L. monocytogenes to breach the blood-brain barrier. The bacterium grows directly in the cytoplasm of infected host cells and moves rapidly throughout and between infected cells using a form of actin-based motility. The L. monocytogenes surface protein, ActA, is expressed in a polarized fashion and interacts with host cell cytoskeletal factors to induce the polymerization of an actin "comet tail" structure that pushes the bacterium through the host cell cytoplasm. The overall goal of this project is to understand the mechanism and biological significance of the actin-based motility of L. monocytogenes. We propose to use an interdisciplinary approach to studying this form of motility at all levels from the biochemistry and biophysics of single molecules involved in the generation of actin-based motility to the dynamics of spread across the blood-brain barrier in infected animals. At the MOLECULAR level, we will determine the minimal functional unit for actin comet tail assembly. At the level of the BACTERIAL CELL, we will establish how ActA localization is determined by bacterial cell wall synthesis and remodeling, and measure the effects of ActA mislocalization on actin comet tail formation and motility. At the level of the HOST CELL, we will identify host cell components that contribute specifically to the process of cell- to-cell spread, and elucidate the possible mechanisms by which bacteria can cross endothelial monolayers. At the level of the HOST ANIMAL, we will determine how the bacteria cross the blood-brain barrier in a newly developed mouse model of systemic infection. Successful completion of our research goals would give significant insight into the mechanisms by which pathogenic bacteria such as L. monocytogenes communicate specifically with the cells of their human hosts to subvert host cell function in favor of bacterial propagation and dissemination. In addition, because L. monocytogenes actin-based motility is a simple model system for force generation by actin polymerization, the results of our research would contribute to our understanding of a wide variety of basic biological processes involving actin-based cell movement, including wound healing, inflammation, embryonic development, and cancer metastasis. PUBLIC HEALTH RELEVANCE: Listeria monocytogenes bacteria cause a rare but very serious form of food poisoning, which can lead to meningitis (infection of the brain and central nervous system) in newborns, immunocompromised people, and older adults, and can also cause late-term miscarriage in pregnant women. These bacteria grow directly inside of human host cells and use their niche inside of human cells to "hide out" from antibodies that the immune system normally generates to fight infections. The short-term goal of this research is to understand how Listeria monocytogenes are able to invade human cells and move from the inside of one human cell to another, with a long-term goal of finding better methods to prevent or treat serious bacterial infections.